SUMMARY

We established that corticotropin-releasing hormone (CRH), CRH-binding
protein (CRH-BP) and CRH-receptor 1 (CRH-R1) are expressed in the gills and
skin of common carp Cyprinus carpio, an early vertebrate.
Immunoreactive CRH was detected in macrophage-like cells in gills and skin, in
fibroblasts in the skin and in endothelial cells in the gills. The involvement
of the CRH system in gills and skin was investigated in response to infection
and in an acute restraint stress paradigm. Carp were infected with the
protozoan leech-transmitted blood flagellate Trypanoplasma borreli
and subjected to acute restraint stress by netting for 24 h. The expression of
CRH-BP and CRH-R1 genes in the gills and in the skin is downregulated after
both infection and restraint. Thus the peripheral CRH system reacts to
infection and stress. The gills and skin separate the internal from the
external environment and are permanently exposed to stress and pathogens.
Because of their pivotal role in maintaining the homeostatic equilibrium,
these organs must act locally to respond to diverse stresses. Clearly, the CRH
system is involved in the response of the integument to diverse stresses at
the vulnerable interface of the internal and external milieu.

Introduction

In fish, in analogy with mammals, the stress response consists of the
activation of the sympathetic nervous system, as well as the
hypothalamus-pituitary-interrenal axis (HPI). In response to hypothalamic
release of corticotropin-releasing hormone (CRH) the pituitary gland increases
the synthesis and release of pro-opiomelanocortin (POMC)-derived peptides
(Wendelaar Bonga, 1997;
Aguilera, 1998;
Dautzenberg and Hauger, 2002).
CRH exerts its effects via specific membrane receptors
(Aguilera et al., 2001). At
least two CRH receptors exist and they differ in their pharmacological
properties and tissue distribution.

CRH expression has been detected in a plethora of mammalian organs
including skin, endometrium, placenta, uterus, ovary, testis, spleen,
pancreas, liver, stomach, small and large intestine, adrenal and thyroid
gland. Furthermore, CRH is produced by various immune cells, including
macrophages (Baker et al.,
2003) as a proinflammatory agent
(Karalis et al., 1997), which
is illustrated by the increased CRH expression during experimentally induced
inflammation (Hargreaves et al.,
1989) or in chronic inflammatory diseases, such as rheumatoid
arthritis (Crofford et al.,
1992,
1993).

The bioactivity of CRH (and related peptides) depends on CRH-binding
protein (CRH-BP), which determines the concentration of bioavailable CRH and
may influence peptide bioactivity and half-life
(Potter et al., 1991;
Seasholtz et al., 2002). As
such CRH-BP may act as a carrier protein that prevents CRH degradation and
facilitates the delivery of peptides to distant sites
(Seasholtz et al., 2002). The
colocalisation of CRH-BP and CRH in both the rostral pars distalis as well as
in the pars intermedia of carp (Huising et
al., 2004) substantiates that CRH-BP is also a regulator of CRH in
the pituitary gland of fish.

Skin and gills in fish are directly and permanently exposed to the
environment and thus to multiple physical, chemical and biological influences.
A direct, local response of skin and gills to pathogens and chemical and
physical stress is envisaged as an important means to guarantee internal
homeostasis.

The fish gill is characterized by an extensive and delicate epithelium that
separates the water from the blood. It is a physiologically diversified organ
that serves respiration, osmoregulation, nitrogen excretion and acid-base
balance, which are key processes that are strongly interrelated. The gill is
the only organ that is perfused by the entire cardiac output and has an
extensive vascular surface area in contact with the plasma
(Olson, 1998); the gills play
a significant, and in some instances dominant, role in endocrine regulation as
an endocrine target as well as metabolically active tissue
(Evans et al., 2005).

The skin protects the fish against injury and infection and is protected by
a chemically and functionally complex mucus coat that is discharged by mucus
cells in the epidermis. It contains a variety of biologically active compounds
including peroxidase (Iger et al.,
1994,
1995;
Brokken et al., 1998), lysozyme
(Rainger and Rowley, 1993),
immunoglobulins, complement and C-reactive protein
(Shephard, 1994).

CRH-BP, CRH-R1 and CRH have been identified in several species of fish,
including carp, in which these proteins have been shown to be involved in the
regulation of the acute stress response
(Huising et al., 2004). To
date, information on peripheral expression of these factors in fish is
limited.

Given the singular importance of fish gills and skin, we investigated the
presence of a local CRH system in these organs. To that end we assessed the
expression of CRH, CRH-BP and CRH-R1 and their messengers by
immunohistochemistry and real-time quantitative PCR in the gill and skin of
carp under normal, stressful and pathological conditions.

Materials and methods

Common carp Cyprinus carpio L. were reared at 23°C in
recirculating UV-treated tapwater at the `De Haar Vissen' facility in
Wageningen, The Netherlands. Fish were fed with pelleted dry food (Provimi,
Rotterdam, The Netherlands) at a daily ration of 0.7% of their estimated body
mass. R3×R8 carp are the offspring of a cross between fish of Polish
origin (R3) and Hungarian origin (R8)
(Irnazarow, 1995). The fish
used in infection experiments were housed in a quarantine unit at Wageningen
University. The restraint experiment was carried out in a purpose-built setup
at the Radboud University, Nijmegen, The Netherlands. At the end of both
experiments, fish were rapidly and irreversibly anaesthetised in the
experimental tanks without prior handling with 0.2 g l-1 tricaine
methane sulphonate (TMS) buffered with 0.4 g l-1 NaHCO3
or with 0.1% 2-phenoxyethanol.

Infection with Trypanoplasma borreli

Three weeks before experiments were started, carp (N=14) were
transferred to a quarantine unit and kept in a single experimental tank. The
R3×R8 carp line is trypanotolerant
(Saeij et al., 2003a). After 3
weeks (t=0) one group (N=8) was injected intramuscularly at
the base of the dorsal fin with 10 000 Trypanoplasma borreli in 100μ
l RPMI and was designated the infected group. The control group
(N=6) was injected with 100 μl RPMI and was marked by a small fin
clip. Three weeks post-infection, when parasitaemia reached peak values
(Saeij et al., 2003b), the
fish were irreversibly anaesthetised. Blood samples were collected for the
determination of haematocrit, leucocrit and parasitaemia.

Restraint period

Two groups of fish (N=8) were housed in identical tanks and after
3 weeks (t=0) one group was restrained for 24 h, bynetting. The other
group did not receive any treatment (controls). Following this 24-h restraint
period, both control and stressed fish were irreversibly anaesthetised. Blood
samples were collected for the determination of haematocrit values as well as
several plasma parameters.

Isolation of head kidney and gill phagocytes

Fish were anaesthetised and blood was collected by puncture of the caudal
vessels. Head kidney and gill phagocytes were isolated as previously described
(Verberg-van Kemenade et al., 1994). Samples of gill and head kidney were
passed through a 50 μm nylon mesh using the barrel from a 10 ml syringe and
suspended in RPMI 1640 medium adjusted to carp osmolarity containing 0.2%
heparin (Leo Pharmaceutical Products, Weesp, The Netherlands). Cell
suspensions were enriched for phagocytes on a 1.07 g cm-3 Percoll
gradient (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The
phagocyte-enriched fraction from the 1.07 Percoll interface was collected and
washed twice with RPMI medium. Viability was assessed by Trypan Blue
exclusion. For RNA isolation 1×107 cells were pelleted and
the RNA was isolated using a RNeasy Mini kit (Qiagen, Valencia, CA, USA)
according to the manufacturer's instructions. For immunohistochemistry the
pelleted cells were fixed in Bouin's solution.

Analysis of CRH, CRH-BP and CRH-R1 gene expression by RQ-PCR

RNA isolation and first strand cDNA synthesis

Gill and skin samples from carp at the end of both infection and restraint
experiments were flash-frozen in liquid nitrogen and stored at -80°C. RNA
was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer's protocol. Single strand cDNA was constructed using Invitrogen
reagents, according to the manufacturer's protocol. Briefly 1 μl 10×
DNAse I reaction buffer and 1 μl DNAse I were added to 1 μg total RNA
and incubated for 15 min at room temperature in a total volume of 10 μl.
DNAse I was inactivated by adding 1 μl 25 mmol l-1 EDTA and
incubated at 65°C for 10 min. To each sample, 300 ng random hexamers, 1μ
l 10 mmol l-1 dNTP mix, 4 μl 5× first Strand buffer, 2μ
l 0.1 mol l-1 dithiotreitol and 10 U RNAse inhibitor were added
and the mixture was incubated for 10 min at room temperature and an additional
2 min at 37°C. Then, 200 U Superscript RNAse H reverse transcriptase (RT)
was added and the reactions were incubated for 50 min at 37°C. A non-RT
control was included for each sample; cDNA was stored at -20°C.

Real time quantitative PCR

The primers for real time quantitative PCR (RQ-PCR) used in this study were
designed and previously used by Huising et al.
(2004). For RQ-PCR 5 μl
cDNA and forward and reverse primers (300 nmol l-1 each) were added
to 12.5 μl Sybr Green PCR Master Mix (Applied Biosystems, Foster City, CA,
USA) and made up with demineralised water to a volume of 25 μl. RQ-PCR (2
min 48°C, 10 min 95°C, 40 cycles of 15 s 95°C and 1 min 60°C)
was carried out on a GeneAmp 5700 Sequence Detection System (Applied
Biosystems). Data were analysed with the ΔΔCt method. Dual
internal standards (40S and β-actin) were incorporated in all RQ-PCR
experiments and results were confirmed to be very similar following
standardisation to either gene. The amplification efficiencies of all primer
sets in this study are very similar and deviate no more than 5% of the optimal
amplification value of 2.0. Only the results standardised for 40S expression
are shown.

Immunohistochemistry

Samples of gill and skin, and the phagocytes isolated from gills and head
kidney were fixed in Bouin's solution, dehydrated in a graded ethanol series
(70, 80, 90, 95 and 100%), embedded in paraffin, cut into 5 μm sections and
mounted on Polysine™-coated slides (Menzel-Glazer®, Braunschweig,
Germany).

Sections were incubated with primary antibodies overnight at 4°C
(CRH-BP) or room temperature (CRH). Goat anti-rabbit IgG-biotin (1:200 Vector
Laboratories, Burlingame, CA, USA) was used as second antibody followed by
amplification using the Vectastain® ABC Amplification Kit (Vector
Laboratories) according to the manufacturer's protocol. AEC
(3-amino-6-ethylcarbazole; Sigma, St Louis, MO, USA) was used as a substrate.
Controls for cross-reactivity of the secondary antibodies and for endogenous
enzyme activity were included in all experiments and were negative. Nuclei
were counterstained with Haematoxylin. After pre-absorption of the primary
antibodies the target cells were negative or in some cases only slightly
positive.

Blood analysis

Blood samples were spun down in a cooled (4°C) microcentrifuge (10 min
at 9500 g, IEC micromax RF; Waldham, MA, USA) and the plasma
was collected and stored at -20°C until use. Cortisol was measured by
radioimmunoassay (RIA) as described previously
(Huising et al., 2004). Plasma
levels of Na+ and K+, glucose, lactate, and the pH were
determined with a Stat Profile®pHOx®Plus (Nova Biomedical, Waldham,
MA, USA, USA).

Cell counting

CRH-BP-positive cells in the gill filaments from the restraint experiment
were quantified in 10 views of the gill filament area using a stereological
overlay method in which a grid was used to estimate the tissue area (in
mm2) (Mazon et al.,
2004) and the results were expressed as number of cells
mm-2.

Statistical analysis

All statistical analyses were carried out with Graphpad Prism software
(3.0). Differences were evaluated with a Student's t-test,
P<0.05 was taken as fiducial limit.

Results

CRH, CRH-BP and CRH-R1 expression at mRNA and protein level

The expression of CRH, CRH-R1 and CRH-BP was studied in the gills and skin
of carp. Expression was plotted relative to the expression of ribosomal
protein 40S. CRH expression was significant but very low in both skin and
gills; CRH-R1 and CRH-BP were expressed more abundantly in both gills and skin
(Fig. 1).

Immunohistochemical studies allowed us to visualise where CRH and CRH-BP
proteins were expressed in the gills and skin. In the gills, macrophage-like
cells, judged by their relatively large volume of cytoplasm and eccentric
nucleus, were positive for CRH or CRH-BP
(Fig. 2A,B). Many cells of
similar appearance were not immunoreactive to either CRH or CRH-BP antiserum.
Other cells that are located within the basal layer in close proximity to the
blood vessel were CRH-positive (Fig.
2A). CRH-BP-positive macrophage-like cells were seen mainly in the
interface between the central venous sinus and the basal layer of the filament
epithelia.

Basal expression of CRH, CRH-R1 and CRH-BP in the gills and skin of C.
carpio. Expression was assessed by RQ-PCR and is plotted relative to the
expression of ribosomal 40S. Values are means ± s.d.
(N=5).

In the skin some of the CRH- and CRH-BP-positive cells had a similar
macrophage-like appearance. CRH-positive cells were observed in the basal
layer of the epidermis, close to the melanocytes and also in the dermis
(Fig. 3A) and in the
fibroblasts of the dermis (Fig.
3B). CRH-BP-positive cells were observed only in the dermis
(Fig. 3C,D).

CRH- and CRH-BP-positive cells were also observed in the phagocytic
fraction from head kidney (not shown) and gills
(Fig. 4A,B). Expression of CRH,
CRH-BP and CRH-R1 was always detectable in the isolated gill phagocytes
(Fig. 4C).

Regulation of CRH-BP and CRH-R1 in gills and skin after infection and
stress

Infection experiment with T. borreli

Three weeks following infection, the number of parasites in the blood was
counted and ranged between 9×106 and 7.5×107
parasites/ml blood in the infected group. There were no parasites present in
the control fish. The values obtained for blood parameters (haematocrit,
leucocrit and plasmatic variables) are listed in
Table 1. The haematocrit was
lower in the infected group than in the control group (P<0.05),
while the leucocrit was higher in the infected group (P<0.05).
There was no significant difference in the mean plasma cortisol levels between
the control and infected group, although cortisol levels fluctuated markedly
between infected, but not control individuals. The lactate concentration in
the plasma was lower in the infected group (P<0.05) than in the
control group. There was no significant difference in the concentration of
Na+, K+, Cl-, glucose and pH between the
control and infected groups.

Blood and plasma parameters from Cyprinus carpio controls and fish 3
weeks into an infection with Trypanoplasma borreli

Expression of CRH-BP and CRH-R1 mRNA in the gills was significantly lower
(P<0.05) in the infected group compared with the non infected
group (Fig. 5A). In the skin a
similar pattern was observed but the effects were not statistically different
(Fig. 5B).

Restraint

The values obtained for blood parameters from the 24 h restraint experiment
are listed in Table 2.
Haematocrit values and the concentration of cortisol and glucose were higher
(P<0.05) in the stressed compared with the control group, while
the Na+ concentration was lower (P<0.05) in the
stressed group. The concentration of K+ and lactate did not differ
between both groups.

Similar to the infection experiment, expression of CRH-BP and CRH-R1 was
lower (P<0.05) in the stressed compared with the control group, in
both gills and skin (Fig. 6).
This reduced gene expression is paralleled by a lower number of
CRH-BP-positive cells in the gills in the stressed than in the control group
(P<0.05; Fig. 6B).
The total number of macrophage-like cells was also lower in the stressed
group, although this difference was not statistically significant. The
expression of ribosomal 40S and α-actin was unaffected by infection or
24 h restraint.

(A) CRH immunoreactive cells and (B) CRH-BP immunoreactive cells in the
phagocytic fraction from C. carpio gills. Arrows indicate the
macrophage-like immunoreactive cells. (C) Expression of CRH, CRH-R1 and CRH-BP
in the phagocytic fraction from carp gills. Expression was assessed by RQ-PCR
and is plotted relative to the expression of ribosomal 40S. Values are means±
s.d. (N=4).

Discussion

We have established that the CRH-BP and CRH-R1 genes are expressed in the
gills and skin of common carp. Expression of their cognate ligand CRH is
detectable in both organs, albeit at a markedly lower level. By
immunohistochemistry it was substantiated that gene expression of CRH and
CRH-BP occurs, at least in part, in macrophage-like cells in the tissue of
gills and skin. Moreover, gene expression of CRH-BP and CRH-R1 markedly drops
in response to acute stress or infection, two different situations that both
involve imminent or ongoing disturbance of homeostasis.

In mammals, the presence of a cutaneous CRH system is well established.
This system responds to diverse stimuli such as immune cytokines, UV radiation
and skin pathology, in which the common denominator appears to be local damage
(Slominski et al., 2000). Yet,
the local effects of CRH in mammalian skin are diverse. CRH inhibits
IL-1α-induced prostaglandin synthesis, presumably via
inhibition of cyclo-oxygenase and phospholipase A2
(Fleisher-Berkovich et al.,
1998). Moreover, direct topic application of CRH to cutaneous or
mucosal tissue evoked vasoconstrictive and anti-inflammatory effects
(Wei and Thomas, 1994;
McLoon and Wirtschafter, 1997;
Gjerde et al., 1998). CRH
injected subcutaneously or intravenously into rats with thermal injury reduced
local fluid accumulation in injured skin of treated animals by over 50%,
independently of the functional activity of the HPI axis (Schafer et al.,
1996,
1997). In contrast to these
studies, which suggest a suppressive effect of CRH on local immune activation,
intradermally injected CRH induces local, CRH-R1-dependent mast cell
degranulation and increases vascular permeability
(Theoharides et al., 1998).
Collectively, these studies indicate that the role of the cutaneous CRH system
is complex, and that the balance between its activation and inhibition may
depend on an interplay with many local as well as systemic parameters.

(A) CRH-R1 and CRH-BP expression levels in gills, (B) the total number of
CRH-BP immunopositive cells and (C) CRH-R1 and CRH-BP expression in skin of
C. carpio following 24 h restraint. Values are means ± s.d.
(N=5). Asterisks indicate significant differences from the controls
(P<0.05). Expression is standardised for ribosomal 40S expression
and the relative quantification value is expressed as
2-ΔΔCt.

Acute restraint stress and infection with a blood parasite had marked
effects on different physiological processes. These physiological changes were
in line with expectations, i.e. changes in leucocrit after infection, and
higher cortisol and glucose levels after stress. In addition we have observed
that the tissues composing the external surfaces of carp express all the major
components of a local CRH system, which is reminiscent of the mammalian skin
CRH system. The general response of this integumental CRH system to stress and
infection is similar. This suggests involvement of diverse local and systemic
signals in the local tissue response. The downregulation of CRH-BP and CRH-R1
in gills and skin in response to acute systemic stress suggests that the
transiently elevated plasma cortisol levels exert a negative feedback on
peripheral CRH-R1 and CRH-BP expression, as was reported earlier for carp
pituitary CRH-R1 expression in the same stress paradigm
(Huising et al., 2004).
Nonetheless, the similar inhibition of gill and skin CRH-BP and CRH-R1
expression observed in the absence of significantly elevated plasma cortisol
levels during T. borreli infection suggests that the expression of
the genes comprising the skin and gill CRH system is locally regulated.
Alternatively, given that CRH and CRH-BP immunoreactivity is at least in part
associated with macrophage-like cells, changes in gene expression may reflect
redistribution of immune cells. Redistribution of immune cells in response to
acute stress (Huising et al.,
2003) or infection with T. borreli
(Scharsack et al., 2003) have
been reported earlier in carp and is considered to contribute significantly to
the local immune surveillance.

In carp gills and skin, expression of CRH is detectable but low, which is
reminiscent of the difficulties in detection of CRH gene expression in the
skin of mice (Slominski et al.,
1996; Slominski and Wortsman,
2000). Although there are ample CRH immunoreactive cells in both
gills and skin, the markedly lower CRH expression compared to the expression
of CRH-BP and CRH-R1 suggests that the local CRH system responds to systemic
CRH or, alternatively, to CRH-related peptides. We previously observed
pronounced immunoreactivity for CRH and CRH-BP in the pituitary pars nervosa
and suggested their involvement in the regulation of the release of one or
several pituitary pars intermedia peptides
(Huising et al., 2004).
Alternatively, systemic CRH from the pituitary pars nervosa may be released
directly into circulation and act on peripheral CRH receptors in gills and
skin. Another possible source of ligand is the caudal neurosecretory system or
urophysis, which contains and releases considerable quantities of urotensin-I,
and, in flounder, has very high expression of CRH
(Lu et al., 2004). Urotensin-I
is a peptide related to CRH that is capable of binding to both CRH-R1 and
CRH-BP (Vaughan et al., 1995;
Behan et al., 1989). Urophysial
urotensin-I was initially discovered for its osmoregulatory capacity, and the
fish gills would form a logical target organ for such signal.

We must also consider the possibility that the local CRH system in gills
and skin can be directly and autonomously activated by an external stress.
Gills and skin are strategically located facing the external and internal
environments, and are permanently exposed to stresses and pathogens. These
factors in combination with the key functions that are united in the fish
integument require a constitutive mechanism to deal with stresses while
cellular/tissue damage is still confined and of low magnitude, i.e. before the
systemic stress response is triggered. The observation of CRH-positive cells
in the basal layer is in line with such a mechanism, as basal layer cells have
previously been reported to protect against high copper exposure from the
water (Dang et al., 1999).

In summary, we have presented evidence for the existence of a local CRH
system in teleost fish. This system is responsive to acute systemic stress as
well as to prolonged infection with the blood flagellate T. borreli.
Therefore, we consider this system analogous to the cutaneous CRH system that
has been reported for human and rodent species. Given the presence of similar
systems in evolutionary distant vertebrate phyla such as fish and mammals
reinforces the importance of a local cutaneous CRH system for the proper and
rapid response to various biological, chemical and physical hazards.

List of abbreviations

CR

corticotropin-releasing hormone

CRH-B

CRH-binding protein

CRH-R

CRH-receptor 1

HP

hypothalamus-pituitary-interrenal axis

POM

pro-opiomelanocortin

RI

radioimmunoassay

RQ-PC

real time quantitative polymerase chain reaction

R

reverse transcriptase

TM

tricaine methane sulphonate

ACKNOWLEDGEMENTS

The present study was realised with support from CNPq, a Brazilian
institute for scientific and technological development and from the Wageningen
Institute of Animal Sciences. We acknowledge Prof. W. W. Vale for providing
the rabbit anti-human CRH-BP antiserum; Dr G. F. Wiegertjes for the advice on
infection with T. borreli, `De Haar Vissen' for excellent animal care
and Tom Spanings for his assistance with the restraint set-up.

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